Three-Dimensional Immunolocalization In Maize Meiocytes

Summary

This immunolocalization protocol in male maize meiocytes maintains the three-dimensional integrity of the cells during meiosis. It allows the dynamic tracking of the intracellular localization of up to three independent antibodies in preserved paraformaldehyde-fixed material, in addition to DAPI, which stains DNA.

Abstract

During meiotic prophase I, the physical configuration of chromosomes varies widely across developmental stages, fortunately these stage-specific configurations allow the proper identification of each of these classical meiotic stages, which are respectively known as leptotene (e.g. the ‘thin threads’), zygotene (e.g. the ‘coupled threads’), pachytene (e.g. the ‘thick threads’), diplotene (e.g. the ‘double threads’) and diakinesis (e.g. the ‘repulsion motion’). The visual analysis of several cytogenetic phenomena that occur during prophase I, such as recombination, pairing, and synapsis, requires highly reliable immunolocalization techniques. In contrast to traditional spreading and squashing techniques that suppress all subcellular and subnuclear information, the 3D conservation procedure described here allows the preservation of key meiotic proteins in their native subcellular location sites (cytoplasmic, nuclear, and subnuclear: euchromatin, heterochromatin, and nucleolar). Thus, this protocol describes how to efficiently fix meiotic anthers, dissect and release meiocytes from anthers, and perform simultaneous immunolocalization of three key proteins in maize male meiocytes.

Introduction

Meiosis is a key process that regulates the transmission of traits in model plants and crops; thus, simple microscopy methods that allow consistent and faithful visualization of protein localization facilitate the understanding of how these traits could be transmitted more efficiently. Unfortunately, the visualization of the localization of proteins inside meiocytes has varied a lot depending on the technique used^1,2. Thus, the aim of the protocol is to provide a straightforward, reproducible method that eliminates artifacts during the observation of meiocytes. For instance, it has long been acknowledged that the observation of meiotic chromosomes is a delicate process that is facilitated by crushing the cells by either simple squashing or by even more disruptive spreading techniques^3,4,5. In Arabidopsis thaliana, researchers commonly employ spreading methods that utilize Carnoy’s fixative (a 3:1 mixture of ethanol and acetic acid) along with various detergents such as Lipsol, Tween-20, and TritonX-100, as well as enzyme mixtures containing cellulase, pectinase, and cytohelicases, to break apart cells and allow for effective immunolocalization on its small chromosomes^1,2. These techniques transform a 3D chromosome conformation into a 2D one, enabling quick observation of the entirety of artificially dispersed chromosomes in a single image. Certain features of chromosomes that endure these harsh treatments may be apparent; however, they often fail to represent the typical behavior of cells and chromosomes in vivo⁶. Quick and very broad detection of up to two antibodies is possible with 2D spreading techniques in various plant species including maize^7,8,9,10,11.

Nonetheless, despite the convenience of legacy 2D techniques, it is now acknowledged that certain phenomena are sensitive to disruptive methods and cannot be observed if the cell, nucleus, or chromosomes are damaged during spreading or squashing. For instance, the movement of the RAD50 protein from the cytoplasm to the nucleus during the leptotene stage, or during centromere coupling (leptonema/zygonema), has been detected only in intact 3D meiocytes¹². Here, readers are offered a detailed 4-day 3D protocol that uses anthers fixed in 4% paraformaldehyde to immunolocalize three key proteins in maize male meiocytes, thereby preserving their structural integrity. Compared to a similar maize 3D immunolocalization methodology⁸, we have added practical notes and troubleshooting tips for researchers. This protocol will benefit the work of plant biologists and plant breeders interested in studying various meiotic processes, such as the control of meiotic recombination by the synaptonemal complex.

Protocol

CAUTION: Handle formaldehyde and acrylamide using appropriate personal protective equipment and a chemical hood; both are toxic (acrylamide is also neurotoxic before polymerization). All the materials used in the study are listed in the Table of Materials.

1. Fixation of maize anthers to preserve the intact structure of meiocytes

1. Use maize plants at the V13 stage with inflorescences still growing inside the stem. During the week of meiotic anther fixation, wear gloves and gently press the enlarged stem at the bump to determine if it’s soft enough to cut open.
   NOTE: This protocol for fixation and immunolocalization may be used for both maize and Arabidopsis thaliana meiotic flower buds (less than 400 µm). But in the case of maize, depending on the variety used and the time of the year (there is quicker growth in spring), it takes around 8 weeks from planting for reference inbred line B73, but only 6 weeks for the early flowering inbred line A344.
2. Preparation of buffers.
   NOTE: The day before chemical fixation, prepare the following working solutions, do it carefully, and label all bottles properly.
   1. Prepare 100 mL of 10x Buffer A (BA) salts, by adding 4.53 g PIPES (Piperazine-N, N′-bis(2-ethanesulfonic acid) at a final concentration of 150 mM), 5.96 g KCl (final concentration 800 mM), 1.17 g NaCl (final concentration 200 mM), 0.74 g EDTA (final concentration 20 mM), 0.19 g EGTA (final concentration 5 mM) and 90 mL H₂O, first adjust pH to 6.8–7 with KOH, then adjust the volume to 100 mL.
   2. Finally, filter sterilize using a disposable Rapid-Flow sterile filter unit with PES of 0.45 µm pore (keep at 4 °C but do not freeze; you may store it for several months).
   3. Prepare:
      -50 mM PIPES (pH adjusted to 7 with KOH).
      - stocks of spermine at a concentration of 0.4 M (previously dissolved in 50 mM PIPES, adjust pH to 7 with KOH, keep aliquots of 50 μL at -20 °C).
      - stocks of spermidine at a concentration of 0.4 M (dissolved in 50 mM PIPES, with pH adjusted to 7 with KOH, keep frozen aliquots of 125 μL at -20°C).
      - stocks of Dithiothreitol (DTT) 1 M (dissolved in 0.01 M sodium acetate, to pH 5.2, keep frozen aliquots of 100 μL at -20 °C).
      - sorbitol 2 M, kept at 4 °C.
3. Prepare 50 mL of 2x Buffer A (BA), on ice, using 10 mL of 10x Buffer A salts (kept at 4 °C), 50 μL of spermine (0.4 M in 50 mM PIPES, with pH adjusted to 7 with KOH), 125 μL of spermidine (0.4 M in 50 mM PIPES, with pH adjusted to 7 with KOH), 100 μL of DTT (1 M in 0.01 M sodium acetate, pH 5.2) vortex vigorously to dissolve; and do not heat.
   1. Then, add 16 mL of 2 M sorbitol. Adjust the final volume to 50 mL with sterile water. Filter-sterilize using a disposable Rapid-Flow sterile filter and store at 4 °C.
4. Prepare a humid chamber made of 5–10 humidified paper towels placed in a plastic box. Verify that the lid seals well. Eliminate any excess water.
5. Then, place in a tray the following items: the humid chamber, a razor blade, fine forceps (previously sterilized with 70% ethanol), a small bottle of sterile water, and masking tape. Bring the tray and its contents to the maize plants checked at step 1.1.
6. Use a razor blade to cut open the bumpy stem at the level of the inflorescence. The main tassel branches, as well as the lateral branches of the inflorescence, can be used for harvesting meiotic florets.
   NOTE: These tassel branches contain the florets of interest to collect meiotic anthers. Each floret contains six anthers, and the 3 upper, larger ones are typically used for analysis. The approximate size and color of the anthers correlate with each meiotic stage. Almost transparent anthers around 1 mm in length are at the leptotene stage, translucent anthers at 1.5 mm in length are at the zygotene stage, whitish anthers at 2 mm in length are at the pachytene stage, and yellowish anthers at > 2 mm in length may contain meiocytes at diplotene, tetrads, and pollen grains that finish gametogenesis. Note that another length-stage correlation can vary among maize genetic backgrounds, and that environmental factors (e.g., high temperature) can accelerate meiotic progression and alter this relationship. It is recommended that researchers validate anther size-stage correlations for each specific maize genotype and growth conditions (via acetocarmine squashing, cf 1.6).
7. Use fine forceps to dissect and carefully collect the lateral branches of the inflorescences and wrap them inside the paper towel of the humid chamber.
   NOTE: With some practice, only a short linear 3–4 cm incision in the stem is needed to extract the branches of the meiotic tassel. You may close this incision by wrapping it three to four times with standard masking tape, which allows you to later harvest the same plant using the remaining undissected inflorescence. Tissue will keep growing and later may be used for pollination.
8. Keep the chamber at room temperature. Use only those branches of the inflorescence that contain florets that carry anthers at the meiotic stage. Make sure to cover the branches with half of the humid towels, but do not immerse them in water.
9. Humidify again if the towels dry out. Close the box until dissection of the anthers, preferably until 30 min before the start of anther collection.
10. Using a binocular stereomicroscope with a calibrated scale, collect fresh anthers from florets into 2 mL of 1x BA in a 35 mm Petri dish, ensuring accurate identification of meiotic stages using the proxy of anther size and color.
    NOTE: To accurately determine the meiocyte stage in sampled anthers, one of three synchronized anthers per floret is quickly analyzed using 3:1 ethanol-acetic acid fixation, acetocarmine staining, and traditional squash microscopy¹³.
11. Prepare the 4% formaldehyde fixative in 1x BA and 0.0025% Tween-20
    NOTE: For 3 mL, add 750 μL formaldehyde 16%, 750 μL H₂O, 1.5 mL 2x BA, and 7.5 μL Tween-20 at 1%.
12. Remove the initial BA buffer and add the 3 mL of this fresh fixative solution to the staged anthers and have them fixated for 45 min inside a new 35 mm Petri dish, sealed by parafilm to prevent formaldehyde evaporation, then place it on a larger Petri dish placed on a rotary shaker (set at 40–60 rpm, or 0.2 x g). Do it at room temperature. Anthers should be swirling slightly for good fixation. They usually float on the surface.
13. Rinse with 3 mL of 1x BA, then transfer the sample to a small Petri dish and place it on a rotary shaker (40–60 rpm) at room temperature for 45 min.
14. Using clean forceps, transfer the anthers to a small 35 mm Petri dish containing fresh 3 mL of 1X BA. Seal the Petri dish with parafilm tightly. Then store the small Petri dish inside a Petri dish, covering it with aluminum foil to keep it in the dark, and place it at 4 °C until use. You may store it for several months if needed.

2. Embedding maize meiocytes within polyacrylamide sandwich pads

DAY 1: 3D slides preparation

1. Prepare 2 mL of acrylamide solution by mixing equal volumes (1:1) of acrylamide and 2x BA. Before adding sodium sulfite (NaS) and ammonium persulfate (APS), degas the acrylamide solution under vacuum for 5–10 min.
2. Aliquot the acrylamide and 2x BA 1:1 ratio mixes by pipetting 100 µL into 1.5 mL tubes.
   The number of tubes you prepare depends on the number of slides you will prepare.
3. Make 1 mL 1x BA.
4. Place 2 cm x 2 cm pieces of parafilm over a Petri dish, then place them under a dissecting microscope. Pipette 50 µL of BA solution with anthers onto parafilm. Trim the pipette tip with scissors for easier anther delivery.
   1. Pipette 25 µL of BA solution onto parafilm elsewhere.
5. Using forceps to hold anthers, cut the tip of the anthers with a scalpel, and gently squeeze the meiocytes out. With a pipette, transfer the meiocytes gently to the 25 µL BA drop and gently mix.
6. Pipette 10 µL from the meiocyte mix onto a separate cover slip.
7. Rapidly, pipette 5 µL of 20% (0.2g/ 1mL) NaS and 5 µL 20% (0.2g/ 1mL) APS into a 1.5 mL tube with Acrylamide/2x Buffer A.
8. Rapidly, pipette 5 µL of the above mix onto a coverslip and mix with meiocytes. Place a poly-L-lysine-capped coverslip on top, but rotate it 45 degrees. Using only one poly-L-lysine coverslip (either commercially obtained or made by immersing coverslips in a poly-L-lysine solution in a Petri dish for a few minutes and letting them dry, on the edge of a new, clean Petri dish) helps the release of a complete pad after polymerization. The coverslip is quickly lifted from the solution and dried before use. For super-resolution microscopy, high-performance coverslips with standardized thickness (≤170 nm) must be used¹⁴.
9. Please do not touch the coverslips for 30–50 min to allow full polymerization of the sample at room temperature (A quicker polymerization process reduces the drying period of the pad, and the resulting quality is less than optimal).
10. Repeat steps ‘2.6–2.9’ for each slide you wish to prepare.
11. Separate all coverslips carefully with a razor blade. Place all coverslips, pad-side up, on a polyacrylamide pad (or most of the pad), in a 6-well plate.
12. Wash each coverslip with a pad with 2 mL of 1x phosphate-buffered saline (PBS) for 10 mins. Repeat this wash once, for a total washing time of 20 min at room temperature.
13. Prepare 50 mL by mixing 49.4 mL PBS 1x, 100 µL EDTA 0.5 M, and 500 µL Triton.
14. Rinse each coverslip with a pad in 1 mL of this 1x PBS supplemented with 1 mM EDTA and 1% Triton for 2 h at room temperature.
15. Prepare a fresh batch of blocking buffer containing 1x PBS, 1 mM EDTA, 0.1% Tween-20, and 3% BSA (1 mL per slide). For 50 mL of blocking buffer, mix 44 mL of PBS 1x, 100 µL of EDTA 0.5 M, 5 mL of 1% Tween-20 solution, and 1.5 g of BSA. BSA is preferred over donkey serum as a blocking agent because it helps minimize non-specific signals when using multiple primary and secondary antibodies.

3. Immunolocalizations

1. Prepare primary antibody (or antibodies) at the wanted dilution using blocking buffer. Combine primary antibodies based on their ability to produce distinct signals when used together. They should come from different organisms, such as rabbit, rat, guinea pig, or chicken, that can be recognized by specific fluorescently labelled secondary antibodies. The use of donkey or goat antibodies is avoided because they are intended for use as secondary antibodies. We recommend using a single species of secondary antibody (e.g., goat in the experiment shown in Figure 1).
2. Add 50 µL of this diluted primary antibody onto the center of each coverslip with a pad.
   Add water between wells to slow down evaporation. Put the plate in a humid chamber (a plastic box with a lid). Leave overnight.

DAY 2: Washing

3. Prepare 500 mL of Wash Buffer (1x PBS + 0.1% Tween-20 + 1 mM EDTA).
4. Remove the overnight solution from the wells by aspiration with your pipette.
5. Quickly wash each coverslip with a pad twice with 1 mL of wash buffer, then remove it from the wells.
6. Pipette another 1 mL/ coverslip with pad and leave sitting for 1 h at room temperature.
   Repeat 7 more times. (8 h total)
7. After the final wash, add water between wells, cover the plate, and leave it overnight in a humid chamber.

DAY 3: Secondary antibody, washes, and mounting slides

8. Prepare the secondary antibodies by diluting each at a ratio of 1:50 in blocking buffer.
   Aspirate the wash solution. Dispense 50 µL of the diluted secondary antibody onto the center of each pad using a pipette.
   NOTE: Every secondary antibody should be used in association with distinct fluorescent dyes with non-overlapping emission wavelengths. For example, one secondary antibody should be labelled with AlexaFluor488 (green), the next one with AlexaFluor555 (orange), and the third with AlexaFluor647 (far red), which can be combined with DAPI (excitation at 461 nM, blue). Other fluorescent dyes, such as DyLight (by Dyomics GmbH), are also usable. In addition, all these secondary antibodies should not cross-react with other species used in the mix of primary antibodies. For example, the anti-rabbit secondary antibody should be cross-adapted to be selective for rabbit antibodies and not recognize either rat or guinea pig antibodies. The same is true for all secondary antibodies. A well-designed experiment should be used to validate all these requirements simultaneously when performing co-immunolocalizations.
9. Add water between the wells, place a plate in the humid chamber, and leave for 2 h at room temperature.
10. Aspirate the solution from the wells.
11. Perform four washes using 1 mL per well, 1 h each at room temperature (4 h total).
12. Wash each well with 1 mL of 1x PBS for 10 min at room temperature and repeat this process two more times. (30 min total)
13. Dilute DAPI in 1x PBS.
14. Stain with a final concentration of 1 µg/mL DAPI (DAPI powder dissolved in water) for 30 min at room temperature, and place 500 µL/well.
15. Remove ProLong™ Gold antifade from the 4 °C freezer to allow it to thaw and reach room temperature.
16. Wash slides in 1x PBS for 30 min at room temperature, with 1 mL/well. Do it twice again. (1 h 30 min total)
17. Remove the last wash.
18. Then add two drops of ProLong™ Gold antifade in the middle of each pad.
19. Mount cover slips on each slide, label every slide, lift the cover slips from the well using fine forceps, and slowly place them on the slide. Using fine forceps, slowly lower a second coverslip onto the first. Using a vacuum, remove excess glycerol from the edges of coverslips. Seal edges of coverslips with two coats of nail polish.
20. If slides are not immediately examined under a fluorescent microscope. Store them in a -20 °C freezer, protected from light, until observation. Later on, let slides sit at room temperature for 1 h in a slide box, horizontally and in the dark, to prevent condensation.

DAY 4: Microscopic observations

21. Observes the slide under the right excitation wavelengths and filters. Standard confocal microscopy is sufficient, but it is advisable to use a microscope with a 60x objective to enable three-dimensional imaging of the entire cell using a z-stack every 0,2 µm, with sequential image acquisitions and deconvolution processing using the Applied Precision SoftWoRx^® software.

Results

Using this protocol, we can simultaneously observe the localization patterns of the three key meiotic proteins in maize (Figure 1). The meiotic protein ABSENCE OF FIRST DIVISION 1 (AFD1) is a homolog of the alpha-Kleisin REC8 subunit of the meiotic cohesin complex. The ASYNAPTIC 1 (ASY1) is a crucial axial element, while the ZYPPER1 (ZYP1) is the transversal element of the synaptonemal complex¹⁵. DAPI staining shows a clear nucleus and chromatin staining, allowing for...

Discussion

This protocol offers a key advantage over others by preserving essential three-dimensional data on meiotic chromosomal protein loading. These features cannot be preserved using regular spreading techniques^1,2. Nonetheless, this protocol has two limitations: it is slow and technically demanding compared to regular spreading techniques performed within one day. In contrast, our protocol requires four days of work, mainly due to extensive washing steps and overnight...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 mL microtubes Axygen	Corning	MCT-150-C
15 mL Falcon tubes	Thermo Fisher Scientific	339651
50 mL Falcon tubes	Thermo Fisher Scientific	339652
6 well plate Falcon polystyrene microplate	Thermo Fisher Scientific	08-772-1B
Acetic acid, 60%	Thermo Fisher Scientific	A38-212
Acrylamide 30%  and bis-acrylamide solution, 29:1	Biorad	1610157
Aluminum fold	Reynolds	U07211R0
Amonium Persulfate (APS)	Merck	A3678
Binocular stereomicroscope	NA	NA
Bovine Serum Albumin (BSA)	Merck	A9418
DAPI	Merck	D9542
DelltaVision microscope	Olympus	NA
Distilled H2O	NA	NA
Disposable Filter Units with PES filters of 0.45 μm pore	Thermo Fisher Scientific/Nalgene	09-740-63B
Fine forceps FST Dumont SS	Fine Science Tools	11200-33
goat (IgG H+L) highly cross-adsorbed  anti-rat labelled by AlexaFluor647	Invitrogen	A-21247
goat (IgG H+L) highly cross-adsorbed anti-rabbit labelled by AlexaFluor568	Invitrogen	A-21428
goat (IgG H+L) highly cross-adsorbed anti-guinea pig labelled by AlexaFluor488	Invitrogen	A-11073
Gloves	Merck	Z412392
Growing chambers / greenhouse / or field	NA	NA
DTT	Merck	D0632
EDTA	Sigma-Aldrich	E6758
EGTA	Sigma-Aldrich	E3889
KCl	Sigma-Aldrich	P3911
Masking tape 3M	Fisher Scientific	19-047-259
Maize seeds	NA
Microscope 22 x 22 mm-1,5 glass coverslips	Fisher Scientific	12-542-B
Microscope 22 x 22 mm-H coverslips	Deckgläser, Zeiss	474030-9020-000
Microscopic glass slides	Fisher Scientific	22-230-900
Microscope slide box	Fisher Scientific,	03-448-9
Micropipets P20, P200, P1000	Merck	CLS4069
NaCl	Merck	S7653
Nail polish	Electron Microscopy Science	72180
Paper towels	Merck	Z188956
Parafilm	Merck	P7668
Paraformaldehyde 16% solution	Electron Microscopy Grade	15710
PBS	Merck	P4474
Plastic box with lid	Tupperware	NA
Poly-L-Lysine	Merck	P4707
Primary antibodies (rat anti-ZmAFD1, rabbit anti-ZmASY1, guinea-pig anti ZmZYP1)	NA
Prolong Antifading	Molecular Probes	P36934
Petri dish (D x H: 35 mm x 10 mm	Corning	351008
Petri dish regular size	Merck	P5731
PIPES	Merck	P1851
Pipet tips (0.5-10 uL) sterile	Merck/Corning	AXYT300
Pipet tips (200 uL) sterile	Merck/Corning	AXYT200Y
Pipet tips (1000 uL) sterile	Merck/Corning	AXYT1000BRS
Razor blade - single edged	Fisher Scientific	12-640
Rocking platform shaker	NA	NA
Spermine	Merck	85590
Spermidine	Merck	S2626
Sorbitol	Merck	S8143
Tween-20	Merck	P9416
Triton X-100	Fisher Scientific	9002-93-1
Ultra pure Acrylamide	Invitrogen	15512023
Vacuum